Methods and compositions for treating platelet-related disorders

ABSTRACT

Provided are prophylactic and therapeutic methods of treatment of subjects for the purpose of inhibiting vaso-occlusive events, including embolism, by administering agents, including anagrelide and anagrelide derivatives, which reduce the number of circulating platelets to low normal to below normal levels. Methods and pharmaceutical preparations comprising such agents are provided.

RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 11/127,544, filed May 11, 2005, to Stephen R. Hanson, entitled“METHODS AND COMPOSITIONS FOR TREATING PLATELET-RELATED DISORDERS,”which is a continuation of U.S. patent application Ser. No. 10/603,401,filed Jun. 25, 2003, to Stephen R. Hanson, entitled “METHODS ANDCOMPOSITIONS FOR TREATING PLATELET-RELATED DISORDERS,” now abandoned,which is a continuation of U.S. patent application Ser. No. 09/666,223,filed Sep. 21, 2000, to Stephen R. Hanson, entitled “METHODS ANDCOMPOSITIONS FOR TREATING PLATELET-RELATED DISORDERS,” now U.S. Pat. No.6,585,995, which issued Jul. 1, 2003, and which claims benefit ofpriority under 35 U.S.C. §119(e) to U.S. provisional application Ser.No. 60/154,929 to Stephen R. Hanson, filed Sep. 21, 1999, entitled“METHODS AND COMPOSITIONS FOR TREATING PLATELET-RELATED DISORDERS.” Thebenefit of priority is claimed to each of these applications. Thedisclosure of each of these applications is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The invention relates to methods and products for treatment and/orprevention of platelet related thrombotic and other vaso-occlusivedisorders.

BACKGROUND OF THE INVENTION

Conditions resulting from thrombotic or thromboembolic events are theleading causes of illness and death in adults in western civilization. Agreat deal of effort and monetary resources have been directed towardsunderstanding the mechanisms involved in vascular occlusive diseasesinvolving thrombotic and thromboembolic events. These efforts haveyielded a number of promising therapeutic agents. Notwithstanding theeffort and financial resources that have been invested, these conditionsstill account for the vast majority of illness and death in the adultpopulations of developed nations.

Platelets are an important cellular component of blood involved inhemostasis as well as thrombotic or thromboembolic events. Abnormallyhigh platelet counts such as those that result from hematologicalproliferative disorders such as for example essential thrombocythemiahave been recognized as an important risk factor in thrombus formation.Furthermore, it has long been accepted that aspirin, which is known toinhibit cyclooxygenase and thereby prevents production of thromboxane A₂in platelets, lowers the incidence of thrombotic and thromboembolicevents. For platelets, therapeutic regimens thus far reported have astheir aim an inhibition of platelet function (e.g., inhibition ofplatelet adhesion, aggregation or factor release). An alternativemodality is the reduction in platelet count in patients with abnormallyhigh levels in certain hematological malignancies to levelsapproximating normal levels. Therapeutic intervention for reducingplatelet count to low normal or below normal levels in subjects withoutmyeloproliferative disorders has not been proposed primarily sincenormal platelet count has been thought to be critical to normalhemostasis.

SUMMARY OF THE INVENTION

The invention in a broad aspect involves the surprising discovery thatsubjects, including those with normal levels of circulating platelets,can unexpectedly derive medical benefit from a reduction in plateletcount to below normal levels, without serious adverse consequences as aresult of the platelet count reduction. The benefit may be proportionalor correlative to the reduction in platelet count in a broad safetyrange. Thus in situations where it is desirable to inhibit apathological condition or process mediated in part by normal levels ofcirculating platelets, subjects can be treated to lower platelet countpreferably to a below normal level, thereby inhibiting the development,progression or propagation of the condition or accelerating or enhancingits regression. The methods of the invention are also useful forreducing the incidence of abnormal vessel growth induced by the presenceof platelets.

A method is provided for treating a subject to reduce the risk ofdeveloping an adverse condition or to inhibit the progression andconsequences of an adverse condition mediated at least in part byplatelets. In some aspects, the subject is treated to reduce plateletcount to low normal levels, while in other aspects the subject istreated to reduce platelet count to below normal levels. In oneembodiment, the subject is treated with a pharmaceutical agent.

In one aspect, the invention provides a method for treating a subject toinhibit a vaso-occlusive event. Inhibiting a vaso-occlusive event meansto prevent the formation of a vaso-occlusive event, to reduceprogression and consequences of an already established vaso-occlusiveevent or to induce regression of a vaso-occlusive event. The inventionalso provides other methods aimed at reducing morbidity or mortality ofsubjects from vaso-occlusive events such as but not limited tothrombotic events which may lead to total or partial vessel blockage bythrombus, or arterial stenosis due to excessive cell proliferation.

The methods of the invention comprise administering to a subject in needof such treatment an agent that reduces platelet count in the subject.The agent is administered in an amount effective to reduce plateletcount in the subject to at least a low normal level. Such reductions inplatelet count will reduce morbidity and/or mortality and therebyprovide patient outcome benefit.

As used herein, a vaso-occlusive event includes a pathological partialocclusion (including a narrowing) or complete occlusion of a bloodvessel, a stent or a vascular graft. A vaso-occlusive event intends toembrace thrombotic and thromboembolic events, and the vascular occlusiondisorders or conditions to which they give rise. Thus, a vaso-occlusiveevent is intended to embrace all vascular occlusive disorders resultingin partial or total vessel occlusion from thrombotic or thromboembolicevents, except those that are related to high platelet count due to ahematological proliferative disorder. A thrombotic event as used hereinis meant to embrace both a local thrombotic event and a distalthrombotic event (e.g., a thromboembolic event such as for example anembolic stroke). A vaso-occlusive event also includes abnormal bloodvessel growth induced by the presence of platelets and the factors theysecrete. An example of this latter form of vaso-occlusive event isintimal hyperplasia which results in a narrowing of the blood vessels(i.e., reduction in the diameter of blood vessels either locally orthroughout an extended segment of the vessel) due to ahyperproliferation of cells of the intimal layer of the blood vesselwall.

Preferably, the subject is otherwise free of symptoms calling fortreatment with the agent. In some embodiments, the subject is preferablyfree of symptoms associated with a hematological proliferative disordersuch as for example myeloproliferative disease. Preferably, the subjectis a human subject, but is not so limited. In another embodiment, thesubject is apparently healthy. In preferred embodiments, the subjects donot have abnormally elevated platelet levels (i.e., a platelet countthat is higher than the normal range) that are caused by a hematologicalproliferative disorder. Thus, preferably, the subjects do not have ahematological proliferative disorder. In an important embodiment, thesubject has a normal platelet count prior to treatment. In someembodiments, the subject has a higher platelet count than the meannormal level but is still considered within the normal range. As anexample, a subject with a platelet count of 450×10³ platelets per μl isconsidered to be at the high end of the normal range and is intended tobe treated by the methods of the invention. In some embodiments, thesubject may have a platelet count above the usual range, but without anyunderlying hematological proliferative disorder. In another embodiment,the subject is not a post-menopausal female.

In some aspects, the invention intends to treat subjects who are at riskof a vaso-occlusive event. These subjects may or may not have had aprevious vaso-occlusive event. The invention embraces the treatment ofsubjects prior to a vaso-occlusive event, at a time of a vaso-occlusiveevent and following a vaso-occlusive event. Thus, as used herein, the“treatment” of a subject is intended to embrace both prophylactic andtherapeutic treatment, and can be used both to limit or to eliminatealtogether the symptoms or the occurrence of a vaso-occlusive event. Inone embodiment, the subject may exhibit symptoms of a vaso-occlusiveevent.

The invention also intends to embrace the treatment of a subject thathas an abnormally elevated risk of a vaso-occlusive event such as athrombotic event. The subject may have vascular disease. The vasculardisease may be selected from the group consisting of arteriosclerosis,cardiovascular disease, cerebrovascular disease, renovascular disease,mesenteric vascular disease, pulmonary vascular disease, ocular vasculardisease or peripheral vascular disease.

In another embodiment, the subject has had a primary vaso-occlusiveevent such as a primary thrombotic event. The agent may be administeredto a subject following a primary vaso-occlusive event. The method of theinvention embraces treatment of a subject to reduce the risk of asecondary thrombotic event or to inhibit the propagation of an existingthrombotic event. The thrombotic event may be selected from the groupconsisting of arterial thrombosis, coronary thrombosis, heart valvethrombosis, coronary stenosis, stent thrombosis and graft thrombosis.The vaso-occlusive event also includes disorders or conditions that mayarise from a thrombotic event or a thromboembolic event and in thisregard a vaso-occlusive event includes but is not limited to myocardialinfarction, stroke and transient ischemic attack. In an importantembodiment the vaso-occlusive event is myocardial infarction. In oneembodiment, the subject has had a myocardial infarction. A subject whohas hypercholesterolemia, hypertension or atherosclerosis also can betreated by the methods of the invention.

In yet another embodiment, the subject is one who will undergo anelective surgical procedure. The agent may be administered to such asubject prior to the elective surgical procedure. The method of theinvention can also be directed towards a subject who has undergone asurgical procedure. As used herein, a surgical procedure is meant toembrace those procedures that have been classically regarded as surgicalprocedures as well as interventional cardiology procedures such asarteriography, angiography, angioplasty and stenting. Thus, the surgicalprocedure, whether elective or not, can be selected from the groupconsisting of coronary angiography, coronary stent placement, coronaryby-pass surgery, carotid artery procedure, peripheral stent placement,vascular grafting, thrombectomy, peripheral vascular surgery, vascularsurgery, organ transplant, artificial heart transplant, vascularangioplasty, vascular laser therapy, vascular replacement, prostheticvalve replacement and vascular stenting.

In a preferred embodiment, the agent is anagrelide. In one embodiment,the agent is a derivative of anagrelide. In important embodiments, theagent is not a 2-aryl benzo[b]thiophene. In other important embodiments,the agent is not raloxifene hydrochloride. However, the agent is not anMPL pathway inhibitory agent (i.e., the agent does not impact upon thesignal transduction pathway involving thrombopoietin and the MPLreceptor).

The agent is administered in an amount effective to reduce plateletcount, in the subject, preferably to at least low normal levels. In oneembodiment, the agent is administered in an amount ranging from 30μg/kg/day to 150 μg/kg/day. In another embodiment, the agent isadministered in an amount ranging from 1 μg/kg/day to 150 μg/kg/day. Insome embodiments, these latter ranges are preferred when the agent isanagrelide or an anagrelide derivative.

In one embodiment, the agent is administered in an amount effective toreduce the platelet count to at least low normal levels if the subjecthas a normal platelet count prior to treatment.

In some embodiments, the agent is administered in an amount effective toreduce the platelet count to below normal levels if the subject has anabove normal platelet count prior to treatment. In these latterembodiments, the subject may not have a hematological proliferativedisorder, but is not so limited.

Depending upon the particular embodiment, the platelet count is reducedanywhere from at least 10% to 95% of pre-treatment levels. In particularembodiments, the platelet count is reduced by at least 90%, at least80%, at least 70%, at least 60%, at least 50%, at least 40%, at least30%, at least 20%, or at least 10%. In some important embodiments, theplatelet count is reduced by more than 10%. In another embodiment,platelet count is reduced by more than 30% or by more than 40%.

In some embodiments that embrace the treatment of a human subject,platelet count is preferably reduced to below 200×10³ platelets per μl,and in still others to below 150×10³ platelets per μl. In still anotherembodiment, platelet count is reduced to below 100×10³ platelets per μlof blood in a human subject. In embodiments in which the platelet countis reduced to a low normal level this is defined as 10% less than themean normal platelet count. In other embodiments, the platelet count isreduced to below normal levels.

In yet another embodiment, the agent is administered in an amounteffective to reduce the platelet count by at least 10% and to an amountabove 200×10³ platelets per μl. In other embodiments, the agent isadministered in an amount effective to reduce the platelet count by atleast 10% and below 200×10³ platelets per ill. In other embodiments, theagent is administered in an amount effective to reduce the plateletcount by at least 20% and to below 200×10³ platelets per μl.

The agent of the invention can be administered simultaneously orconsecutively with another therapeutic compound such as an agent whichwould normally be indicated for the subject. Such agents include agentsfor treating vascular disease or vascular complications (i.e.,complications resulting from such disease). In some importantembodiments, the agent for treating vascular disease or vascularcomplications is an anti-thrombotic agent. The anti-thrombotic agent maybe selected from the group consisting of an anti-coagulant agent, afibrinolytic agent and an inhibitor of platelet function, but is not solimited. Thus, in one embodiment, the agent is administered with aninhibitor of platelet function. The inhibitor of platelet function maybe selected from the group consisting of aspirin, abciximab, clopidogreland dipyridamole. In another embodiment, the agent may be administeredwith an anti-coagulant agent. The anti-coagulant may be selected fromthe group consisting of glycosaminoglycans (e.g., heparins) and vitaminK antagonists. In a further embodiment, the agent is administered with afibrinolytic agent, such as but not limited to one selected from thegroup consisting of plasminogen activators such as tissue plasminogenactivator (TPA), streptokinase and urokinase, plasmin and plasminogen.Depending upon the embodiment, the agent of the invention may beadministered before, simultaneously with or following administration ofthe agent for treating vascular disease or vascular complications.

Other useful categories of such agents include but are not limited toanti-inflammatory agents, anti-thrombotic agents, anti-platelet agents,fibrinolytic agents, lipid reducing agents, direct thrombin inhibitors,glycoprotein IIb/IIIa receptor inhibitors, agents that bind to cellularadhesion molecules and inhibit the ability of white blood cells toattach to such molecules, calcium channel blockers, beta-adrenergicreceptor blockers, cyclooxygenase-2 inhibitors, and angiotensin systeminhibitors.

In one embodiment, the agent is administered following a primaryvaso-occlusive event such as a thrombotic event. The agent can beadministered in a number of ways, including enteral and parenteralroutes. In some preferred embodiments, the agent is administered in asustained release device.

The invention also provides a number of pharmaceutical preparationscomprising agents that reduce platelet count. The pharmaceuticalpreparations of the invention comprise one or more agents that reduceplatelet count and a pharmaceutically acceptable carrier. The agent ispresent in the pharmaceutical preparation in an amount effective toreduce platelet count. In important embodiments of the invention, thepharmaceutical preparation comprises the agent in an amount effective toreduce platelet count to low normal levels or to below normal levels.

In yet a further aspect, the invention provides a sustained releasedevice that comprises an agent that reduces platelet count in a subject,wherein the agent is released for at least 7 days. In one embodiment,the sustained release device further comprises an agent for treatingvascular disease or vascular complications. The agent for treatingvascular disease or vascular complications may be an anti-thromboticagent but is not so limited. In one embodiment, the anti-thrombotic isselected from the group consisting of an anti-coagulant agent, afibrinolytic agent and an inhibitor of platelet function.

Preferably, the agent is released from the sustained release device inan amount effective to reduce platelet count in a subject to low normalor below normal levels. In one embodiment the agent is anagrelide or aderivative of anagrelide. Depending upon the embodiment and the natureof the agent, the sustained release device may release the agent at arate ranging from 30 μg/kg/day to 150 μg/kg/day. In other embodiments,the agent may be released at a rate ranging from 1 μg/kg/day to 150μg/kg/day. In one embodiment, the sustained release device releases theagent for at least 30 days. In other embodiments, the agent is releasedfor at least 6 months, for at least 1 year, for at least 2 years or forat least 5 years or more. Preferably, the agent is released in aneffective amount that does not affect platelet function.

In another aspect, a subject's blood is treated extracorporeally toreduce platelet count to low normal or below normal levels, usingprocedures such as pheresis or adsorption of platelets and removal.Subjects, target platelet count and concurrent therapies are asdescribed above. This aspect of the invention is particularly suited toacute therapy, although it is not so limited.

DETAILED DESCRIPTION OF THE INVENTION

Unexpectedly, the invention involves the discovery that treating humansubjects, especially those who do not have a hematological proliferativedisorder, to induce a lower platelet count (such as to low normal levelsor in some preferred instances to below normal levels) can havedesirable medical benefit without significant adverse side effects.

The invention is premised in part on the discovery that a reduction inplatelet count in a subject, such as for example to low normal and morepreferably below normal levels, reduces the risk of a vaso-occlusiveevent such as a thrombotic event in the subject without significantadverse side effects. As used herein, a vaso-occlusive event is an eventthat is characterized by or results in a decrease in the internaldiameter of blood vessels either locally or systemically to an extentwhich impedes blood flow in a subject and which for the purposes of theinvention is of a pathological nature. Thus, a vaso-occlusive eventembraces pathological narrowing or occlusion of a stent, a vasculargraft or a blood vessel. As used herein, “pathological narrowing orocclusion” refers to narrowing or occlusion which is abnormal and/ordisease-related. A vaso-occlusive event includes events which causeblood vessel narrowing or occlusion (such as thrombotic events,thromboembolic events and intimal hyperplasia) as well as conditionswhich result from such blood vessel narrowing (such as myocardialinfarction and ischemic stroke).

A thrombotic event is an event associated with the formation or presenceof a thrombus in a subject, particularly when present in thevasculature. A thrombus is an aggregation of blood factors, primarilyplatelets and fibrin with entrapment of cellular elements, frequentlycausing vascular obstruction at the point of its formation. Thromboticevents embrace thrombosis at a primary site as well as at a distal site(i.e., thromboembolism). Thrombosis collectively refers to diseasescaused by the formation, development, or presence of a thrombus.Thromboembolism refers to diseases characterized by the blocking of avessel, other than at the initial site of thrombus formation, by athrombus which has been carried to the distal site by the blood current.As used herein, the term thrombosis is intended to embracethromboembolism.

Thrombotic events, including thromboembolic events, can be seriousmedical conditions particularly since they can cause a reduction inblood flow to critical organs including the brain and myocardium.Examples of thrombotic events include but are not limited to arterialthrombosis, including stent and graft thrombosis, cardiac thrombosis,coronary thrombosis, heart valve thrombosis and venous thrombosis.Cardiac thrombosis is thrombosis in the heart. Arterial thrombosis isthrombosis in an artery. Coronary thrombosis is the development of anobstructive thrombus in a coronary artery, often causing sudden death ora myocardial infarction. Venous thrombosis is thrombosis in a vein.Heart valve thrombosis is thrombosis on a heart valve. Stent thrombosisis thrombosis resulting from and/or located in the vicinity of avascular stent. Graft thrombosis is thrombosis resulting from and/orlocated in the vicinity of an implanted graft, particularly a vasculargraft.

Examples of conditions or disorders that result from thrombotic eventsinclude but are not limited to myocardial infarction, stroke, transientischemic attacks, amaurosis fugax, aortic stenosis, cardiac stenosis,coronary stenosis and pulmonary stenosis. Stenosis is the narrowing orstricture of a duct or canal. Coronary stenosis is the narrowing orstricture of a coronary artery. Cardiac stenosis is narrowing ordiminution of any heart passage or cavity. Pulmonary stenosis is thenarrowing of the opening between the pulmonary artery and the rightventricle. Aortic stenosis is narrowing of the aortic orifice of theheart or of the aorta itself.

Vaso-occlusive events also include disorders in which the blood vesselnarrowing results not necessarily from a thrombus but rather athickening of the vessel wall such as with intimal hyperplasia. Intimalhyperplasia refers to a condition characterized by abnormalproliferation of the cells of the intimal layer of the blood vesselwall.

Thus, one aspect of the invention relates to a method for reducing therisk of a thrombotic event. In a particular embodiment, the methodreduces the risk of stroke. Stroke is a condition resulting from thelack of oxygen to the brain, resulting from one or more occlusivethrombi. Depending on the area of the brain affected, stroke can resultin a wide range of symptoms from transient ischemic attacks to death(e.g., coma, reversible or irreversible paralysis, speech problems ordementia). In preferred embodiments, the stroke is non-hemorrhagic innature.

The method of the invention in another embodiment relates to reducingthe risk of myocardial infarction. Myocardial infarction refers to anirreversible injury to the heart muscle. Myocardial infarction generallyresults from an abrupt decrease in coronary blood flow following athrombotic occlusion (e.g., a thromboembolism) of a coronary artery. Thethrombus, in many instances, forms after the rupture of atheroscleroticplaques in diseased coronary arteries. Such injury is highly correlatedwith factors such as cigarette smoking, hypertension and lipidaccumulation.

Transient ischemic attack is a transient acute neurological dysfunctionresulting from a thromboembolism in the cerebral circulation. Amaurosisfugax is the temporary monocular blindness resulting from athromboembolism in the retinal vasculature.

The methods of the invention can be used to reduce the risk of a primaryor a secondary vaso-occlusive event such as a thrombotic event or toinhibit the progression of such an event. A primary vaso-occlusive eventrefers to the first known vaso-occlusive event experienced by thesubject. A secondary vaso-occlusive event refers to a vaso-occlusiveevent which occurs in a subject known or diagnosed as having previouslyexperienced a vaso-occlusive event (i.e., a primary vaso-occlusiveevent).

According to the invention, the risk of a vaso-occlusive event such as athrombotic event is reduced by administering to a subject an agent thatreduces platelet count to a “low normal level” and in some embodimentsto a “below normal level.” A “normal” platelet count as used herein maybe a level in a control population, which preferably includes subjectshaving similar characteristics as the treated individual, such as ageand sex. The “normal” level can also be a range, for example, where apopulation is used to obtain a baseline range for a particular groupinto which the subject falls. Thus, the “normal” value can depend upon aparticular population selected. Preferably, the normal levels are thoseof apparently healthy subjects who have no prior history ofplatelet-mediated disorders. Such “normal” levels then can beestablished as preselected values, taking into account the category inwhich an individual falls. Appropriate ranges and categories can beselected with no more than routine experimentation by those of ordinaryskill in the art. Either the mean or another preselected number withinthe range can be established as the normal preselected value.

As used herein, the terms “platelet level,” “platelet number” and“platelet count” are used interchangeably to refer to the number ofplatelets per a given volume of blood in a subject. The platelet countmay be referred to in a number of ways (e.g., per μl of blood, per ml ofblood, etc.). Generally, platelet counts are referred to herein as thenumber of platelets per μl of blood (i.e., platelets per μl); however,other units may be used.

As is known in the art, the typical range for platelets in a “healthy”human subject is about 150×10³ to 450×10³ platelets per μl of blood(mean 300×10³ platelets per μl). Thus, “below normal levels” ofplatelets, as used herein, in this population is typically less than150×10³ platelets/μl. “Low normal levels” as used herein refer to aplatelet count which is 10% less than the mean normal platelet count.Thus, for the population just mentioned, low normal levels would be270×10³ platelets/μl. Human subjects who have a platelet count of lessthan 100×10³ platelets/μl are considered thrombocytopenic. Plateletcounts of less than 25×10³ platelets/μl indicate severethrombocytopenia. The invention intends to embrace reductions inplatelet counts resulting in platelet counts of equal to or less than270×10³, 260×10³, 250×10³, 240×10³, 230×10³, 220×10³, 210×10³platelets/μl of blood. In preferred embodiments the platelet counts areequal to or less than 200×10³, 190×10³, 180×10³, 170×10³, 160×10³,150×10³, 140×10³, 130×10³, 120×10³, 110×10³, 100×10³ platelets/μl.Nonetheless, it will be understood that it may be desirable depending onfactors such as the particular disease, and the age, and the physicalcondition of the subject that lower levels are desirable, such asplatelet counts equal to or less than 90×10³, 80×10³, 70×10³, 60×10³,50×10³, and 25×10³ platelets/μl of blood. In one embodiment, plateletcount is reduced to below 200×10³ platelets/111 in a human subject. Inmore preferred embodiments, the platelet count is reduced to below150×10³ platelets/μl while in some other even more preferred embodimentsthe platelet count is reduced to below 100×10³ platelets/μl in a humansubject.

In some instances, it may be desirable to treat subjects having aplatelet count in the normal range in order to reduce their plateletcount and thereby reduce the risk of a vaso-occlusive event even if thepost-treatment platelet count is still in the normal range. As anexample, the methods of the invention may be used to treat a subject whohas a platelet count of 450×10³ platelets/μl which while high, is stillin the normal range. The subject may be treated in order to reduce theplatelet count to either a lower level within the normal range (e.g., alow normal level, as described herein) or to a below normal level.

Platelet reductions may also be measured as a percentage of thepre-treatment platelet count in a subject. Thus the agents of theinvention may be administered in an amount effective to reduce plateletcount from at least 10% to at least 95% of pre-treatment levels. In someembodiments, the agents are administered in an amount effective toreduce platelet count by at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90% ofpre-treatment levels. In some embodiments, the subjects are normalsubjects who do not have an abnormally high level of circulatingplatelets such as a platelet count greater than 500×10³ platelets perμl, or greater than 600×10³ platelets per μl which may be due to ahematological proliferative disorder. However, in other embodiments thesubjects are normal subjects who have a high level of circulatingplatelets which is still however within the normal range. The inventionintends to treat this latter group of subjects provided the subject doesnot have a hematological proliferative disorder such asmyeloproliferative disease. In still other embodiments, the subject mayhave a platelet count above the normal range, yet not have ahematological proliferative disorder. In preferred embodiments,platelets are reduced by at least 20% of pre-treatment levels. In morepreferred embodiments, platelets are reduced by at least 20% to at least90% of pre-treatment levels. In still other embodiments, platelets arereduced by over 50% of pre-treatment levels.

The subjects may be treated so as to achieve both a drop in plateletcount below an absolute level (such as for example below 200×10³platelets per μl) and a particular percentage drop in platelet countrelative to pre-treatment levels (such as for example at least 10%). Asan example, a subject may be treated so as to reduce platelet count byat least 20% and to achieve a platelet count of less than 200×10³platelets per μl.

The invention intends to treat subjects who would benefit frominhibiting the growth of an existing thrombus or lowering of the risk ofa vaso-occlusive event such as a thrombotic event. A subject is a mammalincluding humans, nonhuman primates, dogs, cats, sheep, goats, horses,cows, pigs or rodents. The preferred subject is a human. The subject maybe apparently healthy. An apparently healthy subject is one who, at thetime of treatment, does not exhibit disease signs or symptoms. In otherwords, such individuals, if examined by a medical professional, would becharacterized as healthy and free of symptoms of disease. The apparentlyhealthy subjects however may still demonstrate particular risk factorswhich may place them at an elevated risk of a thrombotic event. Forexample, such subjects may be apparently healthy and still have a familyhistory of thrombosis-related disorders. Alternatively, the subject mayhave symptoms of vaso-occlusive disease (such as chest pain, heartpalpitations, shortness or breath, as well as a wide range of othersymptoms well known to a medical practitioner of ordinary skill) or mayhave been diagnosed with such disease.

In still other embodiments the subject is one who is otherwise free ofsymptoms calling for treatment with an agent that reduces platelet countin the subject. These subjects may not necessarily be apparently healthybut at a minimum they do not exhibit symptoms which ordinarily call fortreatment specifically with an agent which reduces platelet count. As anexample, if the agent is anagrelide, the subject can be otherwise freeof signs, symptoms or evidence of disorders for which anagrelide wouldnormally be prescribed (e.g., myeloproliferative disease). Anagrelidehas been prescribed previously for patients diagnosed with essentialthrombocythemia (ET). The hallmark of ET is an abnormally high level ofplatelets in the circulation, generally greater than about 600×10³platelets per μl of blood. Involvement and expansion of otherhemopoietic cell types is not necessarily a common feature of ET.Splenomegaly may also be observed in such patients. Secondarythrombocytosis is another disease state associated with an abnormallyelevated number of platelets. This latter condition is distinguishedfrom ET in that it results from a variety of primary conditions such asrecovery from acute infection, malignant diseases including carcinomaand lymphoma, hemolytic anemia, acute hemorrhage, iron deficiency,response to certain drugs and chronic inflammatory disorders. Subjectsfor whom the methods of the invention are not intended are thosediagnosed with conditions which already call for treatment with an agentsuch as anagrelide, i.e., secondary thrombocytosis, essentialthrombocytosis, polycythemia vera, chronic myelogenous leukemia andmyelofibrosis. In other words, in some preferred embodiments, thesubject is not one who has, or who has been diagnosed with, ahematological proliferative disorder (such as myeloproliferativedisease) which indicates the need for platelet lowering therapy.

The subject can also be one who is at abnormally elevated risk of athrombotic event. The subject to be treated may be one who is prone to athrombotic event. Included in this category of subjects are (1) thosewho have undergone a surgical procedure and are immobilized followingsuch a procedure, (2) those who have chronic congestive heart failure,(3) those who have atherosclerotic vascular disease, (4) those who havemalignancy preferably other than a hematological malignancy whichresults in abnormally high platelet counts, and (5) those who arepregnant. A large majority of human subjects prone to thrombotic eventsdo not manifest any observable perturbation in hemostasis.

One category of subjects with an abnormally elevated risk of athrombotic event is those subjects who have previously experienced aprimary thrombotic event. Subjects having an abnormally elevated risk ofa thrombotic event also include (i) those who have inherited adisposition towards thrombosis, for example those with a family historyof thrombosis related disorders, (ii) those who have acquired a risk ofa thrombotic event such as surgical patients, and (iii) those who engagein lifestyle habits which are considered high risk indicators forthrombosis.

One category of subjects with an abnormally elevated risk of thrombosisis those subjects having vascular disease. Vascular disease is a termwhich broadly encompasses all disorders of blood vessels (collectivelyknown as the vasculature) including small and large arteries and veins,and blood flow. The most prevalent form of vascular disease isarteriosclerosis, a condition associated with the thickening andhardening of the arterial wall. Arteriosclerosis or an arterioscleroticcondition as used herein means classical atherosclerosis, acceleratedatherosclerosis, atherosclerosis lesions and any other arterioscleroticconditions characterized by undesirable endothelial and/or vascularsmooth muscle cell proliferation, including vascular complications ofdiabetes. It is responsible for the majority of deaths in the UnitedStates and in most westernized societies.

Arteriosclerosis of the large vessels is referred to as atherosclerosis.Atherosclerosis is the predominant underlying factor in disorders suchas coronary artery disease, aortic aneurysm, arterial disease of thelower extremities and cerebrovascular disease. Other types ofarteriosclerosis include focal calcific arteriosclerosis (Monckeberg'ssclerosis) and arteriolosclerosis. Arterial diseases other thanarteriosclerosis include congenital structural defects, inflammatory orgranulomatous diseases (e.g., syphilitic aortitis), and small vesseldisorders such as hypertension and autoimmune diseases. Disorders whichare associated with early arteriosclerosis include diabetes mellitus,hypertension, familial hypercholesterolemia, familial combinedhyperlipidemia, familial dysbetalipoproteinemia, familialhypoalphalipoproteinemia, hypothyroidism, cholesterol ester storagedisease, systemic lupus erythrematosus, homocysteinemia, chronic renalinsufficiency, chronic vitamin D intoxication, pseudoxanthoma elasticum,idiopathic arterial calcification in infancy, aortic valvularcalcification in the elderly and Werner's syndrome.

Subjects with cardiovascular disease, cerebrovascular disease and/orperipheral vascular disease (e.g., diabetic feet, failed grafts) arealso considered at abnormally high risk of a thrombotic event.Cardiovascular disease refers to a number of disorders of the heart andvascular system. Cerebrovascular disease refers to a number of disordersof the blood vessels in the cerebrum of the brain. Peripheral vasculardisease encompasses disorders of the peripheral vasculature includingthat of the lower extremities.

The method of the invention can be used to treat subjects at abnormallyelevated risk of experiencing particular vaso-occlusive events. Forexample, a subject with an abnormally elevated risk of myocardialinfarction can be treated according to the method of the invention.Subjects may be treated prophylactically to reduce the risk of a primaryor secondary myocardial infarction. As used herein, subjects having anabnormally elevated risk of myocardial infarction include those withunstable angina, multiple coronary risk factors, and Prinzmetal'svariant angina. Less common etiologic factors includehypercoagulability, coronary emboli, collagen vascular disease, andcocaine abuse.

A subject with an abnormally elevated risk of stroke, for examplenon-hemorrhagic stroke, can also be treated according to the invention.Subjects having an abnormally elevated risk of an ischemic stroke are acategory determined according to conventional medical practice; suchsubjects may also be identified in conventional medical practice ashaving known risk factors for stroke or having increased risk ofcerebrovascular events. The primary risk factors include hypertension,hypercholesterolemia, and smoking. Subjects having an abnormallyelevated risk of an ischemic stroke also include individuals having anycardiac condition that may lead to decreased blood flow to the brain,such as atrial fibrillation, ventrical tachycardia, dilatedcardiomyopathy and other cardiac conditions requiring anticoagulation.Subjects having an abnormally elevated risk of an ischemic stroke alsoinclude individuals having conditions including arteriopathy or brainvasculitis, such as that caused by lupus, congenital diseases of bloodvessels, such as cadasil syndrome, or migraine, especially prolongedepisodes.

Another category of subjects with an abnormally elevated risk of athrombotic event are those subjects who will undergo or those who havealready undergone a surgical or mechanical interventional procedure forthe purposes of vessel repair and/or revascularization. Such proceduresmay be therapeutic or diagnostic in nature, and thus can also beelective or emergency treatments, and most likely involve the risk offormation of thrombi or the release of emboli. Procedures which fallinto this category include but are not limited to vascular surgeryincluding peripheral vascular surgery, vascular grafting, vascular lasertherapy, vascular replacement, including prosthetic valve replacement,and vascular stenting, ventricular assist procedures, artificial hearttransplant, heart and other organ transplants which require aninterfacing of the transplanted organ with the vasculature of thetransplant recipient, thrombectomy, coronary angiography, coronary andperipheral stent placements, carotid artery procedures including carotidendarterectomy, brain angiography, neurosurgical procedures in whichblood vessels are compressed or occluded, cardiac catheterization,vascular angioplasty, including balloon angioplasty, and coronaryby-pass surgery. In addition to the risk of thrombus formation during orimmediately following the surgical procedure, there also exists a riskto subjects who have undergone a surgical procedure and are currentlyimmobilized following the procedure. Thus the invention seeks to embracetreatment of the subject prior to, during and following surgicalprocedures.

Other factors which predispose subjects to abnormally elevated risk of athrombotic event are genetic risk factors and lifestyle habits.Inherited conditions can generally be regarded as hypercoaguable statesor pre-thrombotic states. The pre-thrombotic subject can sometimes beidentified if they present with a personal history of early (i.e.,adolescent or as a young adult) and/or repeated thromboembolic events inthe absence of an overt pre-disposing condition, and/or a family historyof thrombosis related conditions. Subjects who have experienced pain inwalking, ischemia (i.e., a deficiency of blood flow to an area of thebody due to functional constriction or obstruction of a blood vessel),gangrene (i.e., a death of tissue, usually considerable in mass andgenerally associated with loss of blood flow) and chest pain, may beregarded as having a personal history of arterial thrombosis or stroke,and are thus also at risk of a thrombotic event. Risk factors for athrombotic event also include inheritable hematological abnormalitiessuch as deficiency and/or dysfunction in any number of factors includinganti-thrombin III, protein C, protein S and clotting factor V.Cardiovascular abnormalities, i.e., congenital structural abnormalitiesof the cardiovascular system, are also considered risk factors forthrombotic events. Vascular abnormalities such as atherosclerotic plaqueruptures are also considered a risk factor.

Lifestyle risk factors include smoking, failure to exercise and diet tothe extent that it affects other risk factors such as obesity, highcholesterol, hyperlipidemia and high blood pressure (i.e.,hypertension). High cholesterol (i.e., hypercholesterolemia), high bloodpressure (i.e., hypertension), hyperlipidemia, and obesity are mostcertainly also induced by a variety of non-dietary causative elementsincluding genetic and environmental factors.

A hyperlipidemic subject is defined as one whose cholesterol andtriglyceride levels equal or exceed the limits set as described hereinfor both the hypercholesterolemic and hypertriglyceridemic subjects. Ahypercholesterolemic subject (i.e., one with high cholesterol) haseither an LDL (i.e., low-density lipoprotein) level of >160 mg/dL, or anLDL level of >130 mg/dL and at least two risk factors selected from thegroup consisting of male gender, family history of premature coronaryheart disease, cigarette smoking (more than 10 cigarettes per day),hypertension, low HDL (<35 mg/dL), diabetes mellitus, hyperinsulinemia,abdominal obesity, high lipoprotein (a), and a personal history ofcerebrovascular disease or occlusive peripheral vascular disease. Ahypertriglyceridemic subject has a triglyceride (TG) level of >250mg/dL.

Subjects who are hypertensive (i.e., those that have high bloodpressure) are also at risk of a thrombotic event. A hypertensive subjectis one who experiences persistently high arterial blood pressure.Hypertension may have no known cause, in which case it is referred to asessential or idiopathic hypertension. Alternatively, hypertension may beassociated with other primary diseases, in which case it is referred toas secondary hypertension. It is generally considered a risk factor forthe development of heart disease, peripheral vascular disease, strokeand kidney disease. In adults, a diastolic pressure below 85 mmHg isconsidered normal, between 85 and 89 mmHg is considered high normal, 90to 104 mmHg is considered mild hypertension, 105 to 114 mmHg isconsidered moderate hypertension and 115 mmHg or greater is consideredsevere hypertension. When the diastolic pressure is below 90 mmHg, asystolic pressure below 140 mmHg indicates normal blood pressure,between 140 and 159 mmHg is borderline isolated systolic hypertensionand 160 mmHg or higher is isolated systolic hypertension. Thus,generally, normal subjects are those with a blood pressure of 140/90 orless.

Other risk factors which contribute to an elevated risk of thromboticevents, and the disorders which underlie such thrombotic events (e.g.,arteriosclerosis), include hyperlipidemia, hyperglycemia and diabetesmellitus, stress and personality, low index of high density lipoproteins(HDL), male gender, age, hyperinsulinemia, high lipoprotein (a) and apersonal history of cerebrovascular disease or occlusive peripheralvascular disease. Hyperglycemia is a condition associated with too higha level of glucose in the blood, sometimes indicative of uncontrolleddiabetes. It occurs when the body does not have enough insulin or cannoteffectively use insulin to metabolize glucose. This condition may beassociated with diabetes mellitus, Cushing's disease, and Cushing'ssyndrome. Signs of hyperglycemia are significant thirst, dry mouth, andfrequent urination. Normal asymptomatic human subjects who are at least50 years of age, and more preferably 60 years of age, are also atincreased risk for thrombosis.

Subjects at risk of having intimal hyperplasia as well as those havingintimal hyperplasia are also intended to be treated according to themethods of the invention. Thus, the method of the invention can be usedto treat subjects who have or are at risk of having intimal hyperplasia,as well as to reduce the risk of intimal hyperplasia. One common form ofintimal hyperplasia is atherosclerosis.

The invention also intends to treat, in other aspects, subjects who havehad a primary vaso-occlusive event or who are currently experiencing avaso-occlusive event, including subjects who have been diagnosed withthrombosis or as having a thrombotic event. The invention can also beused to treat subjects that manifest an abnormal healing of bloodvessels.

The treatment method of the invention involves the administration to asubject of an agent that reduces circulating platelet count in thesubject. Agents which reduce platelet count are herein sometimesreferred to as platelet reducing agents. Preferably such agents have thespecific effect of reducing only platelet count without affecting levelsof other cell types, although it should be understood that an agent mayalso reduce levels of other cell types provided these latter reductionsdo not induce unacceptable levels of adverse side effects associatedwith such reduction in other cell types. For example, the agent mayreduce levels of megakaryocytes, the precursors of platelets, and suchreduction should not have any undesirable side effect. As anotherexample, an agent may be cytotoxic for a megakaryocyte lineagerestricted cell, such as a platelet, and another blood cell, or a commonprecursor of these two cell types, in which case the agent is acceptableonly if platelet count can be reduced to below normal levels withoutunacceptable levels of side effects associated with such reduction inthe other cell type. In still another example, the agent may inhibitmegakaryocyte function. It will be apparent to persons of ordinary skillin the art how to select and distinguish between such agents.

Agents already known to reduce platelet count include but are notlimited to (1) cAMP phosphodiesterase inhibitors (e.g., anagrelide),6,7-dichloro-1,5-dihydroimidazo-[2,1-b]quinazolin-2(3H)-one or6,7-dichloro-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one (U.S. Pat.Nos. 3,932,407; 4,146,718; RE31,617, Haematologica (1992) 77:40-3), (2)antibodies to cell surface receptors specifically expressed by plateletsor megakaryocytes such as glycoprotein IIb/IIIa receptor antibodies, (3)most chemotherapeutic anti-cancer drugs such as busulphan (Br. J.Haematol. 1986 62:229-37), hydroxyurea (N Engl J Med 1995 332:1132-6),hepsulfan, phosphorus-32 (Br J Radiol 1997 70:1169-73), pipobroman(Scand J. Haematol 1986 37:306-9), cyclophosphamide (J Cell Physiol 1982112:222-8), certain alkylating agents and certain antimetabolites, (4)cytokines, growth factors and interleukins such as alpha-interferon(Cancer Immunol Immunother 1987 25:266-73), gamma-interferon,transforming growth factor-beta, neutrophil activating peptide-2 and itsanalogs (U.S. Pat. No. 5,472,944), macrophage inflammatory protein andits analogs (U.S. Pat. No. 5,306,709), (5) compounds secreted by eitherplatelets or megakaryocytes such as platelet-factor 4 (U.S. Pat. No.5,185,323), transforming growth factor-beta, the 12-17 kD glycoproteinproduced by megakaryocytes, thrombin and thrombospondin and its amino(1-174 amino acid) terminal fragment (J Lab Clin Med 1997 129:231-8),and (6) other agents including anti-cheloid agents such as Tranilast(Rizaben) (J Dermatol 1998 25:706-9); forskolin and spleenanti-maturation factor (U.S. Pat. No. 4,088,753).

All the aforementioned agents may be suitable for use in the method ofthe invention to reduce normal platelet count in a subject preferably toa below normal level with the purpose of preventing or treating avaso-occlusive event such as a thrombosis. In some instances thesebenefits are achieved by reducing the platelet count to low normal whilein other more preferred instances the platelet count is reduced to belownormal levels. It should be understood that the agents useful in theinvention may be capable of affecting platelet function as well asreducing platelet count. However, preferably, such agents are used in adose, formulation and administration schedule which favor the plateletcount reducing activity of the agent and do not impact significantly, ifat all, on platelet function.

Another category of agents which reduce platelet counts is MPL pathwayinhibitory agents. However, the invention does not intend to embracethis category of agent in the methods provided herein.

A preferred agent is anagrelide. Although anagrelide is capable ofaffecting platelet function, it is used in the compositions and methodsof the invention in a dose, formulation and administration schedulewhich reduces platelet count (preferably to below normal levels) withoutsignificantly impacting upon platelet function. Analogs (e.g.,derivatives) of anagrelide which are as effective or more effective thanthe parent compound are also intended for use in the method of theinvention. Preferably, such analogs would also be screened for anincreased potency and specificity towards the megakaryocyte lineage withlimited side effects. Synthesis of anagrelide analogs can beaccomplished through routine chemical modification methods such as thoseroutinely practiced in the art. Analogs of anagrelide have been reportedby a number of groups. Jones et al. reported the synthesis of an analog,RS-82856(N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo-[2,1b]quinazolin-2-one(J. Med. Chem. 1987 30:295-303). Other inhibitors of platelet CAMP20phosphodiesterases synthesized by directed replacement of side chains onanagrelide have been reported by Meanwell et al. (J. Med. Chem. 199235:2672-87). Other anagrclide analogs have been documented in U.S. Pat.Nos. 3,932,407; 4,146,718 and RE31,617. For example, Beverung, Jr. etal. (US RE31,617 (1984)) discloses optionally substituted1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-ones and6(H)-1,2,3,4-tetrahydropyrimido[2,1-b]quinazolin-2-ones. These include1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-nitro-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-amino-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,6-hydroxy-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-hydroxy-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,8-bromo-6-[H]-1,2,3,4-tetrahydroimidazo[2,1-b]-quinazolin-2-one,6-methyl-7-nitro-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,7-bromo-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-chloro-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,6-chloro-7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,6,7-dichloro-1,2,3,5-tetrahydro-imidazo[2,1-b]-quinazolin-2-one,7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,3-(carboxymethyl)-3,4-dihydro-5-methyl-4-methylene-1H-quinazolin-2-one,3-(carboxymethyl)-4,5-dimethyl-1,2,3,4-tetrahydroquinazoline-2-one,2-chloro-3-carethoxymethyl-4,5-dimethyl-3,4-dihydroquinazoline,5,6-dimethyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,3-(carbethoxymethyl)-3,4-dihydro-6-methylene-1H-quinazolin-2-one,3-(carbethoxymethyl)-4,6-dimethyl-1,2,3,4-tetrahydroqionazolin-2-one,2-chloro-3-carbethoxymethyl-4,6-dimethyl-3,4-dihydroquinazoline,5,7-dimethyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one,5-methyl-3-(carbethyoxymethyl)-1,2,3,4-tetrahydroquinazolin-2-one,2-chloro-3-carbethoxymethyl-5-methyl-3,4-dihydroquinazolinehydrochloride and6-methyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one.

Many of the above-listed agents while capable of reducing platelet countcan also impact upon other cell lineages, particularly other hemopoieticcell lineages. It is preferred that the agents used in the methods ofthe invention are provided or administered in a manner which limitseffects on other cell types. One way this can be accomplished is toidentify agents which while perhaps not exclusive for the megakaryocytelineage, have limited specificity for other cell lineages. Most of theagents listed above fall into this category. Another way of limitingunnecessary side effects is to administer the agent in the maximal dosewhich reduces platelet count and which does not impact upon other celltypes. Such determination can be made using in vitro clonogenic assayssuch as those described herein, which are standard in the art. Yetanother way of providing specificity is to conjugate an otherwisenon-specific agent with a molecule which will target the non-specificagent to megakaryocytes and platelets. Potential targeting molecules arethose which normally bind receptors uniquely expressed on cells of themegakaryocyte lineage.

The methods of the invention utilize this library technology to identifysmall molecules including small peptides which bind to receptor ligandbinding sites. One advantage of using libraries for antagonistidentification is the facile manipulation of millions of differentputative candidates of small size in small reaction volumes (i.e., insynthesis and screening reactions). Another advantage of libraries isthe ability to synthesize antagonists which might not otherwise beattainable using naturally occurring sources, particularly in the caseof non-peptide moieties.

Methods for preparing libraries of molecules are well known in the artand many libraries are commercially available. Libraries of interest inthe invention include synthetic organic combinatorial libraries.Libraries are also meant to include, but are not limited to, syntheticsmall molecule libraries and chemical libraries. The libraries can alsocomprise cyclic carbon or heterocyclic structure and/or aromatic orpolyaromatic structures substituted with one or more of theabove-identified functional groups.

Small molecule combinatorial libraries may also be generated. Acombinatorial library of small organic compounds is a collection ofclosely related analogs that differ from each other in one or morepoints of diversity and are synthesized by organic techniques usingmulti-step processes. Combinatorial libraries include a vast number ofsmall organic compounds. One type of combinatorial library is preparedby means of parallel synthesis methods to produce a compound array. A“compound array” as used herein is a collection of compoundsidentifiable by their spatial addresses in Cartesian coordinates andarranged such that each compound has a common molecular core and one ormore variable structural diversity elements. The compounds in such acompound array are produced in parallel in separate reaction vessels,with each compound identified and tracked by its spatial address.Examples of parallel synthesis mixtures and parallel synthesis methodsare provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and itscorresponding PCT published patent application WO95/18972, publishedJul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and itscorresponding PCT published patent application WO96/22529, which arehereby incorporated by reference.

One way of testing putatively useful agents is to perform in vitroassays in which platelets or platelet precursors (e.g., megakaryocytes,or megakaryocyte precursors) are exposed to a compound after which theirmorphology (for example using an appropriate cell staining techniquesuch as Wright's stain), number (for example using a Coulter counter)and/or colony forming ability are tested. These latter assays can beperformed using either cell lines known to differentiate into themegakaryocyte lineage, or to the megakaryocyte lineage, several of whichhave been established in the prior art and examples of which include theBa/F3 and UT-7/GM cell lines, or primary hemopoietic tissue, such asbone marrow. The number and quality of megakaryocyte colonies can bedetermined as a function of the presence and absence of the librarymember. Preferably, the assays are carried out by culturing the cells ina semi-solid culture in an amount of thrombopoietin sufficient tostimulate maximal megakaryocyte colony growth from the cell population.The library member is then titrated into the cultures in order todetermine the amount necessary to reduce megakaryocyte colony formation.In this manner, in addition to the amount of antagonist necessary toinhibit megakaryocyte growth altogether, one can also determine thatamount which inhibits the growth by a particular percentage. Forexample, if it desirable to reduce megakaryocyte growth andproliferation by 50% in order to achieve a reduction in platelet countin vivo, then the assay can be used to determine that amount ofantagonist necessary to inhibit megakaryocyte colony growth by 50%. Animportant benefit of a clonogenic assay, such as that described herein,is the ability to analyze the effect of the library member on a widevariety of hemopoietic cell types. Since it is possible, with a correctcocktail of growth factors, to stimulate the growth of a variety ofhemopoietic lineages in culture, the effect of the library member oneach lineage can be studied. Thus, library members can be furtherscreened for their selective action on the megakaryoctye lineage.Clonogenic assays such as those described herein are routinely employedby artisans of ordinary skill. Each of the aforementioned in vitroscreening assays is amenable to high-throughput screening.

Another way of measuring the biological activity of the syntheticcompound is to perform in vivo assays in which animals, preferably mice,are injected, for example intravenously, with the compound and thenanalyzed for megakaryocyte growth and proliferation or plateletproduction. Hemopoietic populations, such as bone marrow and spleen, canbe harvested from treated animals and plated into in vitro semi-solidclonogenic cultures in order to determine the effect of the librarymember on megakaryocytes. Preferably the number and quality ofmegakaryocyte colonies derived from test animals should be compared tothat of animals injected with control carrier (e.g., saline).Alternatively, animals can be assayed directly for platelet counts. Thiscan be done in a number of ways including by bleeding the animals(usually from the tail or retro-orbital vein) and counting the number ofplatelets either manually using a hemocytometer or through the use of anautomated cell counter, such as a Coulter counter. Adverse side effectscan also be tested in animals injected with putative antagonists in thismanner. One possible adverse side effect may be an inability to clot dueto a severe reduction in platelets. To assess clotting function,standard bleeding assays can be employed which measure the time requiredfor bleeding from an experimentally induced wound to clot and thus stop.Platelet count and bleeding assays are routinely performed in humansubjects as a measure of platelet count and platelet activity. Humansubjects with a platelet count of more than 100×10³ platelets per μl ofblood are generally asymptomatic and their bleeding times are within thenormal range. Bleeding times of less than 10 minutes are considerednormal. When platelet count falls below 100×10³ platelets per μl, thebleeding time is extended and appears to be linearly related to theplatelet count. Human subjects with a platelet count of less than 50×10³platelets per μl experience easy bruising, while those with a plateletcount of less than 20×10³ platelets per μl are prone to spontaneousinternal bleeding. Platelet count and bleeding assays are routinelypracticed by those of ordinary skill in the art and are taught inHarrison's Principles of Internal Medicine, Isselbacher, McGraw Hill,New York (1994).

Physical methods also exist for reducing platelet count. These methodsinclude platelet-pheresis which is the centrifugal separation ofplatelets from other blood cellular components. Platelet-pheresisprovides the benefit of effecting platelet reduction in a short periodof time. This may be desirable for a subject unexpectedly scheduled foran elective surgery. Other physical methods for reducing platelet countinvolve the use of adsorption of platelets onto solid state matricescoated with binding partners specific for platelets. As an example,platelets may be removed from blood using positive selection affinityfiltration. Such an approach may involve applying peripheral blood to acolumn containing a solid matrix to which is coupled a growth factorsuch as thrombopoietin. Another example of affinity chromatography mayinvolve elution of blood over an affinity matrix, having as the solidstate Sephadex G-10, coated with fibrinogen, fibronectin, or preferably,an Arg-Gly-Asp tripeptide, all of which are known to bind platelets.This latter approach has been reported by Besselink et al. for bindingof human platelets. (J. Biomater. Sci. Polym. Ed. (1995) 7:551-562)Other binding partners for platelets which could be used in anadsorption technique to separate platelets from blood are glycoproteinIIb/IIIa antagonists such as Ro-43-8857 and L-700,462 reported by Cooket al. (Thromb. Haemost. (1993) 70:838-47). Yet another form of affinityseparation useful in the removal of platelets from blood is immuneaffinity which uses a solid matrix coupled to an antibody specific forplatelets and/or megakaryocytes such as an anti-glycoprotein IIb/IIIareceptor, or a fragment thereof.

Thus, in this aspect of the invention, a subject's blood is removed,depleted of platelets, and then returned until overall platelet count isbelow normal.

The invention provides pharmaceutical preparations of the agents of theinvention. These pharmaceutical preparations comprise the agent of theinvention and also a pharmaceutically acceptable carrier. Thepharmaceutical preparations may be administered in effective amounts.The effective amount will depend upon the mode of administration, theparticular condition being treated and the desired outcome. It will alsodepend upon, as discussed above, the stage of the condition, the age andphysical condition of the subject, the nature of concurrent therapy, ifany, and like factors well known to the medical practitioner. Forprophylactic applications, it is that amount sufficient to delay theonset of, inhibit the progression of, or halt altogether the particularcondition being treated, thereby producing patient benefit. Fortherapeutic applications, it is that amount sufficient to achieve amedically desirable result, thereby producing patient benefit. In someinstances, patient benefit may be measured by a reduction in morbidityand/or mortality. In some cases this is a decrease in cell maturationand/or proliferation. In the case of megakaryocytes, the medicallydesirable result may be to inhibit thrombosis via blocking ofmegakaryoctye maturation, endoreduplication and/or proliferation. Inother cases, it is an increase in platelet consumption, elimination ordeath. Ultimately, the amount which is administered is one effective forreducing platelet count to low normal and more preferably below normallevels in a subject, without a significant level of adverse sideeffects.

Generally, doses of active compounds of the present invention would befrom about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected thatdoses ranging from 1-500 mg/kg, and preferably doses ranging from 1-100mg/kg, and even more preferably doses ranging from 1-50 mg/kg, will besuitable. In most preferred embodiments, the agents will be administeredin doses ranging from 1 μg/kg/day to 10 mg/kg/day, with even morepreferred doses ranging from 1 μg/kg/day to 0.150 μg/kg/day and from 30μg/kg/day to 150 μg/kg/day depending upon the purpose of theintervention and the subject to be treated. The latter dose range ispreferred if the agent is anagrelide. A variety of administration routesare available. The methods of the invention, generally speaking, may bepracticed using any mode of administration that is medically acceptable,meaning any enteral or parenteral mode that produces effective levels ofthe active compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intrapulmonary, intracavitary, transdermal, intradermal,transmucosal, subcutaneous, intravenous, intraarterial, intramuscular,or local routes. The term “parenteral” includes subcutaneous,intravenous, intramuscular, or infusion. Injectable routes such asintravenous or intramuscular routes are not particularly suitable forlong-term therapy and prophylaxis. They could, however, be preferred insituations where oral administration is contra-indicated. Oraladministration will be preferred for prophylactic or therapeutictreatment because of the convenience to the patient as well as thedosing schedule.

Compositions suitable for oral administration may be presented asdiscrete units, in both immediate release or controlled releaseformulations such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions as well as injectabledrug delivery devices such as controlled release preparations. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

The agents that reduce platelet count may be combined, optionally, witha pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the agents of the present invention, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

Other delivery systems can include immediate release or controlledrelease formulations. Examples of controlled release formulationsinclude time-release, delayed release or sustained release deliverysystems. Such systems can reduce toxicity, increase efficacy and avoidrepeated administrations of the platelet reducing agent, reducingpeak-related side effects and increasing convenience to the subject andthe physician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include but are notlimited to polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, lipids, polyesteramides,polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.Microcapsules of the foregoing polymers containing drugs are describedin, for example, U.S. Pat. No. 5,075,109, and non-polymer systems suchas melted and recrystallized sterols including cholesterol. Deliverysystems also include non-polymer systems that are: lipids includingsterols such a cholesterol, cholesterol esters and fatty acids orneutral fats such as mono- di- and tri-glycerides; hydrogel releasesystems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the platelet reducing agentis contained in a form within a matrix such as those described in U.S.Pat. Nos. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systemsin which an active component permeates at a controlled rate from apolymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and5,407,686. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

For example, U.S. Pat. No. 4,452,775 discloses that the relativeproportions of active agent and matrix components can be varied withindefined ranges depending on the macromolecule to be administered and thedesired rate and duration of release. The macromolecular active agentmay comprise 0.1-10.0%, by weight, of the delivery system. The actualamount of active agent incorporated in the delivery system will dependon the particular active agent, the desired effect, and, to a limitedextent, the desired duration of release; a preferred amount of activeagent is 2 to 5%, by weight, of the system. The relative proportions ofthe matrix components may be varied within the following ranges:

(a) 20-80% cholesterol powder;

(b) 20-80% cholesterol prills, 100-1200 microns in diameter;

(c) 0.1-5.0% biocompatible binding agent;

(d) 0.1-5.0% biocompatible lubricating agent.

A preferred matrix composition comprises, by weight:

(a) 40-60% cholesterol powder;

(b) 40-60% cholesterol prills having diameters of 200-800 microns;

-   (c) 0.1-1.0% biocompatible binding agent; and

(d) 0.5-1.5% biocompatible lubricating agent.

An especially preferred matrix composition comprises, by weight:

(a) 48-52% cholesterol powder;

(b) 48-52% cholesterol prills having diameters of 420-710 microns;

-   (c) 0.4-0.6% biocompatible binding agent; and

(d) 0.8-1.0% biocompatible lubricating agent.

The size of the cholesterol prill may vary from about 100-1200 micronsin diameter. A preferred range is about 400-700 microns. The choices ofmatrix component proportions and cholesterol prill size are governed inlarge part by the macromolecule to be delivered, the desired releaserate and duration of release, and the body site at which the system isto be placed. Those skilled in the art can readily determine the rate ofdiffusion of active agents through the cholesterol matrix when wetted bythe biological fluids of the targeted body site, and adjust the ratiosand sizes of matrix components to achieve a delivery system which isbest suited to the particular active agent and site of prills, binder,lubricant and active agent for a particular application.

U.S. Pat. No. 4,452,775 discloses that a number of binders andlubricating agents are known. The binding agent used may be chosen fromamong, for example, the U.S.P. grade polyethylene glycols havingmolecular weights between 1,250 (PEG 1250) and 8,000 (PEG 8000),polyvinylpyrollidone, hydroxymethylcellulose,hydroxypropylmethylcellulose, and the materials commercially known underthe trademark Pluronic™ with molecular weights between 3,800 (Pluronic™L101) and 14,000 (Pluronic™ F127). A preferred binding agent is PEG6000. Depending on the method of manufacture, incorporation in thematrix of an effective amount of a lubricating agent may facilitateproduction. Suitable lubricants include for example, stearic acid,magnesium stearate, calcium stearate, or Sterotex™. Preferred amongthese are stearic acid and magnesium stearate. The foregoing lists ofbinders and lubricants are not intended to be exhaustive of thematerials which are compatible with the scope and intention of thisinvention, but merely set out examples to illustrate the types oflubricants and binders which may be used.

Use of a long-term sustained release implant (or device) may beparticularly suitable for treatment of subjects at elevated risk of avaso-occlusive event such as one resulting from a thrombotic event.These subjects would include subjects scheduled for elective vascularsurgery. Long-term release, as used herein, means that the implant isconstructed and arranged to deliver levels of the active ingredient forat least 1 week, in some instances for at least 30 days, and in othersfor at least 60 days. In some aspects of the invention that involvelonger-term treatment and prevention, it is desirable that the sustainedrelease device release effective amounts of agent for at least 6 months,I year, 2 years or in some cases, 5 years or more. Long-term sustainedrelease implants are well-known to those of ordinary skill in the artand include some of the release systems described above.

Regardless of the particular agent used in the methods of the invention,administration via a sustained release device is preferable in someinstances because it can reduce the peak levels of agent which are oftenobserved with single bolus administrations (such as non-continuousinjection or oral delivery). A reduction in the peak level of agent inthe subject also reduces the likelihood of side effects. As an example,a side effect resulting from the ingestion of anagrelide is diarrhea.Sustained release of anagrelide would minimize this side effect.

The agent of the invention should be administered for a length of timesufficient to provide either or both therapeutic and prophylacticbenefit to the subject. Generally, the agent is administered for atleast one day. In some instances, particularly where a subject has had avaso-occlusive event or where the subject is at risk of such an event,the agent may be administered for the remainder of the subject's life.The rate at which the agent is administered may vary depending upon theneeds of the subject and the mode of administration. For example, it maybe necessary in some instances to administer higher and more frequentdoses of the agent to a subject for example during or immediatelyfollowing a vaso-occlusive event (such as a myocardial infarction),provided still that such doses reduce platelet count but do notsignificantly affect platelet function. On the other hand, it may bedesirable to administer lower doses in order to maintain a desiredplatelet count once it is achieved. In still other embodiments, the samedose of agent may be administered throughout the treatment period whichas described herein may extend throughout the lifetime of the subject.The frequency of administration may vary depending upon thecharacteristics of the subject. The agent may be administered daily,every 2 days, every 3 days, every 4 days, every 5 days, every week,every 10 days, every 2 weeks, every month, or more, or any timetherebetween as if such time was explicitly recited herein.

In other aspects, the agents of the invention are administered withanother agent, preferably an agent that would normally be indicated forthe subject. In some embodiments, the agents may be administeredsubstantially simultaneously with the other therapeutic agents. Bysubstantially simultaneously, it is meant that a platelet reducing agentof the invention is administered to a subject close enough in time withthe administration of the other therapeutic agent, whereby the twocompounds may exert an additive or even synergistic effect, e.g.,reducing platelet function by decreasing platelet count and inhibitingtheir ability to aggregate. In other embodiments, the platelet reducingagents of the invention can be administered before or after theadministration of the other therapeutic agent.

The agents of the invention may be administered with several categoriesof therapeutic agents, although preferably these agents are those whichwould normally be indicated for the subject. Generally, these agents arethose which are useful and which are currently indicated for treatingvascular disorders and vascular complications. These agents can beclassified in terms of their function or in terms of the disorders forwhich they are indicated. Several useful categories of such agentsinclude but are not limited to anti-inflammatory agents, anti-thromboticagents, anti-platelet agents, fibrinolytic agents, lipid reducingagents, direct thrombin inhibitors, glycoprotein IIb/IIIa receptorinhibitors, agents that bind to cellular adhesion molecules and inhibitthe ability of white blood cells to attach to such molecules, calciumchannel blockers, beta-adrenergic receptor blockers, cyclooxygenase-2inhibitors, and angiotensin system inhibitors.

One broad category of agents which may be administered with the plateletreducing agents of the invention is anti-thrombotic agents.Anti-thrombotic agents are defined as agents which prevent the formationof a blood thrombus via a number of potential mechanisms and theyinclude fibrinolytic agents, anti-coagulant agents and inhibitors ofplatelet function.

Fibrinolytic agents are defined as agent that lyse a thrombus (e.g., ablood clot), usually through the dissolution of fibrin by enzymaticaction. Examples of thrombolytic agents include but are not limited toancrod, anistreplase, bisobrin lactate, brinolase, Hageman factor (i.e.factor XII) fragments, molsidomine, plasminogen activators such asstreptokinase, tissue plasminogen activators (TPA) and urokinase, andplasmin and plasminogen. Anti-coagulant agents also include inhibitorsof factor Xa, factor TFPI, factor VIIa, factor IXc, factor Va, factorVIIIa as well as inhibitors of other coagulation factors.

Anti-coagulant agents are agents which inhibit the coagulation pathwayby impacting negatively upon the production, deposition, cleavage and/oractivation of factors essential in the formation of a blood clot.Anti-coagulant agents include but are not limited to vitamin Kantagonists such as coumarin and coumarin derivatives (e.g., warfarinsodium); glycosoamino-glycans such as heparins both in unfractionatedform and in low molecular weight form; ardeparin sodium, bivalirudin,bromindione, coumarin dalteparin sodium, desirudin, dicumarol, lyapolatesodium, nafamostat mesylate, phenprocoumon, sulfatide, and tinzaparinsodium.

Other “anti-coagulant” and/or “fibrinolytic” agents include plasminogen(to plasmin via interactions of prekallikrein, kininogens, Factors XII,XIIIa, plasminogen proactivator, and tissue plasminogen activator[TPA]);streptokinase; urokinase: anisoylated plasminogen-streptokinaseactivator complex; pro-urokinase; (Pro-UK); rTPA (alteplase or activase;r denotes recombinant); rPro-UK; abbokinase; eminase; streptase;anagrelide hydrochloride; bivalirudin; dalteparin sodium; danaparoidsodium; dazoxiben hydrochloride; efegatran sulfate; enoxaparin sodium;ifetroban; ifetroban sodium; tinzaparin sodium; retaplase; trifenagrel;warfarin; and dextrans.

Still other anti-coagulant agents include, but are not limited to,ancrod; anti-coagulant citrate dextrose solution; anticoagulant citratephosphate dextrose adenine solution; anticoagulant citrate phosphatedextrose solution; anticoagulant heparin solution; anticoagulant sodiumcitrate solution; ardeparin sodium; bivalirudin; bromindione; dalteparinsodium; desirudin; dicumarol; heparin calcium; heparin sodium; lyapolatesodium; nafamostat mesylate; phenprocoumon; tinzaparin sodium; warfarinsodium.

Heparin may stabilize symptoms in evolving stroke, but anticoagulantsare useless (and possibly dangerous) in acute completed stroke, and arecontraindicated in hypertensives because of the increased possibility ofhemorrhage into the brain or other organs. Although the timing iscontroversial, anticoagulants may be started to prevent recurrentcardiogenic emboli. Clot lysing agents, including tissue plasminogenactivator and streptokinase, are being evaluated for the very earlytreatment of acute stroke. Nimodipine has recently been shown to improvesurvival and clinical outcome after ischemic stroke.

Inhibitors of platelet function are agents that impair the ability ofmature platelets to perform their normal physiological roles (i.e.,their normal function). Platelets are normally involved in a number ofphysiological processes such as adhesion, for example, to cellular andnon-cellular entities, aggregation, for example, for the purpose offorming a blood clot, and release of factors such as growth factors(e.g., platelet-derived growth factor (PDGF)) and platelet granularcomponents. One subcategory of platelet function inhibitors areinhibitors of platelet aggregation which are compounds which reduce orhalt the ability of platelets to associate physically with themselves orwith other cellular and non-cellular components, thereby precluding theability of a platelet to initiate the formation of a thrombus.

Examples of useful inhibitors of platelet function include but are notlimited to acadesine, anagrelide (if given at doses exceeding 10mg/day), anipamil, argatroban, aspirin, clopidogrel, cyclooxygenaseinhibitors such as nonsteroidal anti-inflammatory drugs and thesynthetic compound FR-122047, danaparoid sodium, dazoxibenhydrochloride, diadenosine 5′,5′″-P1,P4-tetraphosphate (Ap4A) analogs,difibrotide, dilazep dihydrochloride, 1,2- and 1,3-glyceryl dinitrate,dipyridamole, dopamine and 3-methoxytyramine, efegatran sulfate,enoxaparin sodium, glucagon, glycoprotein IIb/IIIa antagonists such asRo-43-8857 and L-700,462, ifetroban, ifetroban sodium, iloprost,isocarbacyclin methyl ester, isosorbide-5-mononitrate, itazigrel,ketanserin and BM-13.177, lamifiban, lifarizine, molsidomine,nifedipine, oxagrelate, prostaglandin E (PGE), platelet activatingfactor antagonists such as lexipafant, prostacyclin (PGI2), pyrazines,pyridinol carbamate, ReoPro (i.e., abciximab), sulfinpyrazone, syntheticcompounds BN-50727, BN-52021, CV-4151, E-55 10, FK-409, GU-7, KB-2796,KBT-3022, KC-404, KF-4939, OP-41483-, TRK-100, TA-3090, TFC-612 andZK-36374, 2,4,5,7-tetrathia-octane, 2,4,5,7-tetrathiaoctane 2,2-dioxide,2,4,5-trithiahexane, theophyllin, pentoxifyllin, thromboxane andthromboxane synthetase inhibitors such as picotamide and sulotroban,ticlopidine, tirofiban, trapidil and triclopidine, trifenagrel,trilinolein, 3-substituted 5,6-bis(4-methoxyphenyl)-1,2,4-triazines,antibodies to glycoprotein IIb/IIIa as well as those disclosed in U.S.Pat. No. 5,440,020, anti-serotonin drugs, dipyridamole, clofibrate,caffeine and ticlopidine.

“Anti-inflammatory” agents include alclofenac; alclometasonedipropionate; algestone acetonide; alpha amylase; amcinafal; amcinafide;amfenac sodium; amiprilose hydrochloride; anakinra; anirolac;anitrazafen; apazone; balsalazide disodium; bendazac; benoxaprofen;benzydamine hydrochloride; bromelains; broperamole; budesonide;carprofen; cicloprofen; cintazone; cliprofen; clobetasol propionate;clobetasone butyrate; clopirac; cloticasone propionate; cormethasoneacetate; cortodoxone; deflazacort; desonide; desoximetasone;dexamethasone dipropionate; diclofenac potassium; diclofenac sodium;diflorasone diacetate; diflumidone sodium; diflunisal; difluprednate;diftalone; dimethyl sulfoxide; drocinonide; endrysone; enlimomab;enolicam sodium; epirizole; etodolac; etofenamate; felbinac; fenamole;fenbufen; fenclofenac; fenclorac; fendosal; fenpipalone; fentiazac;flazalone; fluazacort; flufenamic acid; flumizole; flunisolide acetate;flunixin; flunixin meglumine; fluocortin butyl; fluorometholone acetate;fluquazone; flurbiprofen; fluretofen; fluticasone propionate;furaprofen; furobufen; halcinonide; halobetasol propionate; halopredoneacetate; ibufenac; ibuprofen; ibuprofen aluminum; ibuprofen piconol;ilonidap; indomethacin; indomethacin sodium; indoprofen; indoxole;intrazole; isoflupredone acetate; isoxepac; isoxicam; ketoprofen;lofemizole hydrochloride; lomoxicam; loteprednol etabonate;meclofenamate-sodium; meclofenamic acid; meclorisone dibutyrate;mefenamic acid; mesalamine; meseclazone; methylprednisolone suleptanate;morniflumate; nabumetone; naproxen; naproxen sodium; naproxol; nimazone;olsalazine sodium; orgotein; orpanoxin; oxaprozin; oxyphenbutazone;paranyline hydrochloride; pentosan polysulfate sodium; phenbutazonesodium glycerate; pirfenidone; piroxicam; piroxicam cinnamate; piroxicamolamine; pirprofen; prednazate; prifelone; prodolic acid; proquazone;proxazole; proxazole citrate; rimexolone; romazarit; salcolex;salnacedin; salsalate; salycilates; sanguinarium chloride; seclazone;sermetacin; sudoxicam; sulindac; suprofen; talmetacin; talniflumate;talosalate; tebufelone; tenidap; tenidap sodium; tenoxicam; tesicam;tesimide; tetrydamine; tiopinac; tixocortol pivalate; tolmetin; tolmetinsodium; triclonide; triflumidate; zidometacin; glucocorticoids; andzomepirac sodium. One preferred anti-inflammatory agent is aspirin.

“Lipid reducing” agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, probucol, lovastatin, fluvastatin, simvastatin,atorvastatin, pravastatin, cirivastatin.

“Direct thrombin inhibitors” include hirudin, hirugen, hirulog,agatroban, PPACK, thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” are both antibodies andnon-antibodies, and include but are not limited to ReoPro (abcixamab),lamifiban, tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v.52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogeneous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, aminone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexylene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenyloin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,medroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, sotalol, nadolol, tiprenolol, tomalol, timolol, bupranolol,penbutolol, trimepranol,2-(3-(1,1-dimethyl-ethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrileHCl, 1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)-phenoxy)-2- propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxypropyl-thio)-4-(5-carbamoyl-2-thienyl)thiazol,and 7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, anti-inflammatory drugs exert most oftheir anti-inflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/H synthase and/or prostaglandin-endoperoxide synthase). Initially,only one form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It was originally identified in bovine seminalvesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (See, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from COX-1. COX-2 is rapidly andreadily inducible by a number of agents including mitogens, endotoxin,hormones, cytokines and growth factors. As prostaglandins have bothphysiological and pathological roles, the constitutive enzyme, COX-1, isresponsible, in large part, for endogenous basal release ofprostaglandins and hence is important in their physiological functionssuch as the maintenance of gastrointestinal integrity and renal bloodflow. By contrast, it is believed that the inducible form, COX-2, ismainly responsible for the pathological effects of prostaglandins whererapid induction of the enzyme would occur in response to such agents asinflammatory agents, hormones, growth factors, and cytokines. Therefore,it is believed that a selective inhibitor of COX-2 has similaranti-inflammatory, antipyretic and analgesic properties to aconventional non-steroidal anti-inflammatory drug, and in additioninhibits hormone-induced uterine contractions and also has potentialanti-cancer effects, but with reduced side effects. In particular, suchCOX-2 inhibitors are believed to have a reduced potential forgastrointestinal toxicity, a reduced potential for renal side effects, areduced effect on bleeding times and possibly a decreased potential toinduce asthma attacks in aspirin-sensitive asthmatic subjects, and aretherefore useful according to the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as COX-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. Pat. No.5,604,253 “N-benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif.). Additional COX-2 inhibitors are alsodescribed in U.S. Pat. No. 5,643,933, assigned to G. D. Searle & Co.(Skokie, Ill.), entitled: “Substituted sulfonylphenyl-heterocycles ascyclooxygenase-2 and 5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, a pseudoglobulin in blood plasma,to produce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II(angiotensin-[1-8]octapeptide). The latter is an active pressorsubstance which has been implicated as a causative agent in severalforms of hypertension in various mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs.Among the most potent and useful renin-angiotensin system inhibitors arerenin inhibitors, ACE inhibitors, and angiotensin II antagonists. In apreferred embodiment of the invention, the renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹)(Val⁵)(Ala⁸)]angiotensin-(1-8) octapeptide and related analogs);N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634); imidazoleacetate derivatives including 2-N-butyl-4-chloro-1-(2-chlorobenzile)imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.247(1), 1-7 (1988)); 4, 5, 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid and analogderivatives (U.S. Pat. No. 4,816,463); N2-tetrazole beta-glucuronideanalogs (U.S. Pat. No. 5,085,992); substituted pyrroles, pyrazoles, andtryazoles (U.S. Pat. No. 5,081,127); phenol and heterocyclic derivativessuch as 1,3-imidazoles (U.S. Pat. No. 5,073,566); imidazo-fused 7-memberring heterocycles (U.S. Pat. No. 5,064,825); peptides (e.g., U.S. Pat.No. 4,772,684); antibodies to angiotensin II (e.g., U.S. Pat. No.4,302,386); and aralkyl imidazole compounds such as biphenyl-methylsubstituted imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(−1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclohexa-pentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566 (E-alpha-2-[2-butyl-1-(carboxyphenyl) methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, Pa.); Losartan (DUP753/MK954,DuPont Merck Pharmaceutical Company); Remikirin (RO42-5892, F. HoffmanLaRoche AG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G. D. Searle and Company).

“Angiotensin converting enzyme” (ACE), is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di- andtri-peptides and antibodies to ACE which intervene in therenin-angiotensin system by inhibiting the activity of ACE therebyreducing or eliminating the formation of pressor substance angiotensinII. ACE inhibitors have been used medically to treat hypertension,congestive heart failure, myocardial infarction and renal disease.Classes of compounds known to be useful as ACE inhibitors includeacylmercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat.No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyldipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S.Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S.Pat. No. 4,587,258), and perindopril (U.S. Pat. No. 4,508,729),carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No.4,512,924) and benazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoylprolines such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di- andtri-peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies (see earlier discussion on antibodies).

Other than aspirin, ticlopidine is another antiplatelet agent that hasbeen shown to be beneficial for stroke treatment. Endarterectomy may beindicated in patients with 70 to 99 percent narrowing of a symptomaticinternal carotid artery. However, most authorities agree that carotidendarterectomy is not indicated in patients with TIAs that are referableto the basilar-vertebral system, in patients with significant deficitsfrom prior strokes, or in patients in whom a stroke is evolving.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-COA to mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-COA reductase inhibitors hasbeen used to treat individuals with hypercholesterolemia. More recently,HMG-CoA reductase inhibitors have been shown to be beneficial in thetreatment of stroke (Endres M, et al., Proc Natl Acad Sci USA, 1998,95:8880-5).

HMG-CoA reductase inhibitors useful for co-administration with theagents of the invention include, but are not limited to, simvastatin(U.S. Pat. No. 4,444,784), lovastatin (U.S. Pat. No. 4,231,938),pravastatin sodium (U.S. Pat. No. 4,346,227), fluvastatin (U.S. Pat. No.4,739,073), atorvastatin (U.S. Pat. No. 5,273,995), cerivastatin, andnumerous others described in U.S. Pat. No. 5,622,985, U.S. Pat. No.5,135,935, U.S. Pat. No. 5,356,896, U.S. Pat. No. 4,920,109, U.S. Pat.No. 5,286,895, U.S. Pat. No. 5,262,435, U.S. Pat. No. 5,260,332, U.S.Pat. No. 5,317,031, U.S. Pat. No. 5,283,256, U.S. Pat. No. 5,256,689,U.S. Pat. No. 5,182,298, U.S. Pat. No. 5,369,125, U.S. Pat. No.5,302,604, U.S. Pat. No. 5,166,171, U.S. Pat. No. 5,202,327, U.S. Pat.No. 5,276,021, U.S. Pat. No. 5,196,440, U.S. Pat. No. 5,091,386, U.S.Pat. No. 5,091,378, U.S. Pat. No. 4,904,646, U.S. Pat. No. 5,385,932,U.S. Pat. No. 5,250,435, U.S. Pat. No. 5,132,312, U.S. Pat. No.5,130,306, U.S. Pat. No. 5,116,870, U.S. Pat. No. 5,112,857, U.S. Pat.No. 5,102,911, U.S. Pat. No. 5,098,931, U.S. Pat. No. 5,081,136, U.S.Pat. No. 5,025,000, U.S. Pat. No. 5,021,453, U.S. Pat. No. 5,017,716,U.S. Pat. No. 5,001,144, U.S. Pat. No. 5,001,128, U.S. Pat. No.4,997,837, U.S. Pat. No. 4,996,234, U.S. Pat. No. 4,994,494, U.S. Pat.No. 4,992,429, U.S. Pat. No. 4,970,231, U.S. Pat. No. 4,968,693, U.S.Pat. No. 4,963,538, U.S. Pat. No. 4,957,940, U.S. Pat. No. 4,950,675,U.S. Pat. No. 4,946,864, U.S. Pat. No. 4,946,860, U.S. Pat. No.4,940,800, U.S. Pat. No. 4,940,727, U.S. Pat. No. 4,939,143, U.S. Pat.No. 4,929,620, U.S. Pat. No. 4,923,861, U.S. Pat. No. 4,906,657, U.S.Pat. No. 4,906,624 and U.S. Pat. No. 4,897,402, the disclosures of whichpatents are incorporated herein by reference.

Nitric oxide (NO) has been recognized as a messenger molecule with manyphysiologic roles, in the cardiovascular, neurologic and immune systems(Griffith, T M et al., J Am Coll Cardiol, 1988, 12:797-806). It mediatesblood vessel relaxation, neurotransmission and pathogen suppression. NOis produced from the guanidino nitrogen of L-arginine by NO synthase(Moncada, S and Higgs, E A, Eur J Clin Invest, 1991, 21:361-374). Agentsthat upregulate endothelial cell Nitric Oxide Synthase include, but arenot limited to, L-arginine, rho GTPase function inhibitors (seeInternational Application WO 99/47153, the disclosure of which isincorporated herein by reference), and agents that disrupt actincytoskeletal organization (see International Application WO 00/03746,the disclosure of which is incorporated herein by reference).

“Co-administering,” as used herein, refers to administeringsimultaneously two or more compounds of the invention (e.g., anagrelide,and an agent known to be beneficial in the treatment of, for example, acardiovascular condition e.g., an anticoagulant), as an admixture in asingle composition, or sequentially, close enough in time so that thecompounds may exert an additive or even synergistic effect, i.e., onreducing cardiomyocyte cell-death in a cardiovascular condition.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. It is intended that the inventionencompass all such modifications within the scope of the appendedclaims.

All references, patents and patent applications and publications thatare cited or referred to in this application are incorporated in theirentirety herein by reference.

1. A method for treating a subject to inhibit a vaso-occlusive event,comprising administering anagrelide or a derivative or analog thereof toa subject in need of such treatment, wherein the anagrelide orderivative or analog thereof is administered in an amount effective toreduce platelet count in the subject to at least a low normal level. 2.The method of claim 1, wherein: the anagrelide or a derivative or analogthereof is provided in a controlled release delivery system; and thedelivery system is formulated to administer an amount of the anagrelideor the anagrelide derivative or analog at a rate and in an amounteffective to reduce platelet count in the subject to at least a lownormal level but without significantly affecting platelet function. 3.The method of claim 1, wherein the subject does not have a hematologicalproliferative disorder.
 4. The method of claim 2, wherein the subjectdoes not have a hematological proliferative disorder
 5. The method ofclaim 1, wherein the subject has a normal platelet count prior totreatment.
 6. The method of claim 1, wherein the subject has an abovenormal platelet count prior to treatment.
 7. The method of claim 1,wherein the subject is a human.
 8. The method of claim 1, wherein thesubject has vascular disease.
 9. The method of claim 8, wherein thevascular disease is selected from the group consisting ofarteriosclerosis, cardiovascular disease, cerebrovascular disease,renovascular disease, mesenteric vascular disease, pulmonary vasculardisease, ocular vascular disease and peripheral vascular disease. 10.The method of claim 1, wherein the subject has had a primaryvaso-occlusive event.
 11. The method of claim 1, wherein the subject hasa condition selected from the group consisting of hypercholesterolemia,hypertension and atherosclerosis.
 12. The method of claim 1, wherein theanagrelide or derivative or analog thereof is administered to thesubject prior to a surgical procedure.
 13. The method of claim 12,wherein the surgical procedure is selected from the group consisting ofcoronary angiography, coronary stent placement, coronary by-passsurgery, carotid artery procedure, peripheral stent placement, vasculargrafting, thrombectomy, peripheral vascular surgery, vascular surgery,organ transplant, artificial heart transplant, vascular angioplasty,vascular laser therapy, vascular replacement and vascular stenting. 14.The method of claim 1, wherein the anagrelide or derivative or analogthereof is administered to the subject after a surgical procedure. 15.The method of claim 14, wherein the surgical procedure is selected fromthe group consisting of coronary angiography, coronary stent placement,coronary by-pass surgery, carotid artery procedure, peripheral stentplacement, vascular grafting, thrombectomy, peripheral vascular surgery,vascular surgery, organ transplant, artificial heart transplant,vascular angioplasty, vascular laser therapy, vascular replacement andvascular stenting.
 16. The method of claim 2, wherein the controlledrelease delivery system further comprises another therapeutic compoundselected from among an inhibitor of platelet function, an anti-coagulantagent and a fibrinolytic agent.
 17. The method of claim 16, wherein theinhibitor of platelet function is selected from among acadesine,anipamil, argatroban, aspirin, clopidogrel, a cyclooxygenase inhibitor,a nonsteroidal anti-inflammatory drug, the synthetic compound FR-122047,danaparoid sodium, dazoxiben hydrochloride, a diadenosine5′,5′″-P1,P4-tetraphosphate (Ap4A) analog, difibrotide, dilazepdihydrochloride, 1,2-glyceryl dinitrate, 1,3-glyceryl dinitrate,dipyridamole, dopamine, 3-methoxytyramine, efegatran sulfate, enoxaparinsodium, glucagon, a glycoprotein IIb/IIIa antagonist, Ro-43-8857,L-700,462, ifetroban, ifetroban sodium, iloprost, isocarbacyclin methylester, isosorbide-5-mononitrate, itazigrel, ketanserin, BM-13.177,lamifiban, lifarizine, molsidomine, nifedipine, oxagrelate,prostaglandin E (PGE), a platelet activating factor antagonist,lexipafant, prostacyclin (PGI2), a pyrazine, pyridinol carbamate,abciximab, sulfinpyrazone, BN-50727, BN-52021, CV-4151, E-5510, FK-409,GU-7, KB-2796, KBT-3022, KC-404, KF-4939, OP-41483, TRK-100, TA-3090,TFC-612, ZK-36374, 2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane2,2-dioxide, 2,4,5-trithiahexane, theophyllin, pentoxifyllin, athromboxane inhibitor, a thromboxane synthetase inhibitor, picotamide,sulotroban, ticlopidine, tirofiban, trapidil, triclopidine, trifenagrel,trilinolein, a 3-substituted 5,6-bis(4-methoxyphenyl)-1,2,4-triazine, anantibody to glycoprotein IIb/IIIa, an anti-serotonin drug, dipyridamole,clofibrate, caffeine and ticlopidine.
 18. The method of claim 17,wherein the inhibitor of platelet function is selected from the groupconsisting of aspirin, abciximab, clopidogrel and dipyridamole.
 19. Themethod of claim 2, wherein the controlled release delivery systemcomprises a matrix in which the anagrelide or the anagrelide derivativeor analog is dispersed.
 20. The method of claim 19, wherein the matrixis formulated to achieve a reduction in peak level of agent in thesubject.
 21. The method of claim 2, wherein the delivery systemcomprises one or more than one polymer selected from among apoly(lactide-glycolide), a copolyoxalate, a polycaprolactone, apolyesteramide, a polyorthoester, a polyhydroxybutyric acid or apolyanhydride.
 22. The method of claim 2, wherein the delivery systemcomprises a lipid, a sterol, a fatty acid, a neutral fat, a wax or acombination thereof.
 23. The method of claim 2, wherein the deliverysystem comprises a hydrogel delivery system.
 24. The method of claim 2,wherein the delivery system comprises microcapsules.
 25. The method ofclaim 24, wherein the microcapsules comprise one or more than onepolymer selected from among a poly(lactide-glycolide), a copolyoxalate,a polycaprolactone, a polyesteramide, a polyorthoester, apolyhydroxybutyric acid or a polyanhydride.
 26. The method of claim 2,wherein the delivery system is formulated for administration by a routeselected from among oral, rectal, topical, nasal, intradermal,intramuscular and parenteral routes.
 27. The method of claim 1, whereinplatelet count is reduced by at least 20%.
 28. The method of claim 1,wherein platelet count is reduced by at least 50%.
 29. The method ofclaim 1, wherein platelet count is reduced to below 200×10³ plateletsper μl.
 30. The method of claim 1, wherein platelet count is reduced tobelow 150×10³ platelets per μl.
 31. The method of claim 1, whereinplatelet count is reduced to below 100×10³ platelets per μl.
 32. Themethod of claim 1, wherein platelet count is reduced by at least 10% andto an amount above 200×10³ platelets per μl.
 33. The method of claim 1,wherein platelet count is reduced by at least 10% and to an amount below200×10³ platelets per μl.